Macrophage migration inhibitory factor (MIF) is a cytokine initially isolated based upon its ability to inhibit the in vitro random migration of peritoneal exudate cells from tuberculin hypersensitive guinea pigs (containing macrophages) (Bloom et al. Science 1966, 153, 80-2; David et al. PNAS 1966, 56, 72-7). Today, MIF is known as a critical upstream regulator of the innate and acquired immune response that exerts a pleiotropic spectrum of activities.
The human MIF cDNA was cloned in 1989 (Weiser et al., PNAS 1989, 86, 7522-6), and its genomic localization was mapped to chromosome 22. The product of the human MIF gene is a protein with 114 amino acids (after cleavage of the N-terminal methionine) and an apparent molecular mass of about 12.5 kDa. MIF has no significant sequence homology to any other protein. The protein crystallizes as a trimer of identical subunits. Each monomer contains two antiparallel alpha-helices that pack against a four-stranded beta-sheet. The monomer has additional two beta-strands that interact with the beta-sheets of adjacent subunits to form the interface between monomers. The three subunits are arranged to form a barrel containing a solvent-accessible channel that runs through the center of the protein along a molecular three-fold axis (Sun et al. PNAS 1996, 93, 5191-5196).
It was reported that MIF secretion from macrophages was induced at very low concentrations of glucocorticoids (Calandra et al. Nature 1995, 377, 68-71). However, MIF also counter-regulates the effects of glucocorticoids and stimulates the secretion of other cytokines such as tumor necrosis factor TNF-α and interleukin IL-1β (Baugh et al., Crit. Care Med 2002, 30, S27-35). MIF was also shown e.g. to exhibit pro-angiogenic, pro-proliferative and anti-apoptotic properties, thereby promoting tumor cell growth (Mitchell, R. A., Cellular Signalling, 2004. 16(1): p. 13-19; Lue, H. et al., Oncogene 2007. 26(35): p. 5046-59). It is also e.g. directly associated with the growth of lymphoma, melanoma, and colon cancer (Nishihira et al. J Interferon Cytokine Res. 2000, 20:751-62).
MIF is a mediator of many pathologic conditions and thus associated with a variety of diseases including inter alia inflammatory bowel disease (IBD), rheumatoid arthritis (RA), acute respiratory distress syndrome (ARDS), asthma, glomerulonephritis, IgA nephropathy, myocardial infarction (MI), sepsis and cancer, though not limited thereto.
Polyclonal and monoclonal anti-MIF antibodies have been developed against recombinant human MIF (Shimizu et al., FEBS Lett. 1996; 381, 199-202; Kawaguchi et al, Leukoc. Biol. 1986, 39, 223-232, and Weiser et al., Cell. Immunol. 1985, 90, 16778).
Anti-MIF antibodies have been suggested for therapeutic use. Calandra et al., (J. Inflamm. 1995. 47, 39-51) reportedly used anti-MIF antibodies to protect animals from experimentally induced gram-negative and gram-positive septic shock. Anti-MIF antibodies were suggested as a means of therapy to modulate cytokine production in septic shock and other inflammatory disease states.
U.S. Pat. No. 6,645,493 discloses monoclonal anti-MIF antibodies derived from hybridoma cells, which neutralize the biological activity of MIF. It could be shown in an animal model that these mouse-derived anti-MIF antibodies had a beneficial effect in the treatment of endotoxin induced shock.
US 200310235584 discloses methods of preparing high affinity antibodies to MIF in animals in which the MIF gene has been homozygously knocked-out.
Glycosylation-inhibiting factor (GIF) is a protein described by Galat et al. (Eur. J. Biochem, 1994, 224, 417-21). MIF and GIF are now recognized to be identical. Watarai et al. (PNAS 2000, 97, 13251-6) described polyclonal antibodies binding to different GIF epitopes to identify the biochemical nature of the posttranslational modification of GIF in Ts cells.
In view of the clear biological significance of MIF/GIF, is therefore necessary and would be highly desirable to provide purified anti-MIF antibodies as diagnostic and therapeutic tools.
Clearly, therefore a need exists for the production of anti-MIF antibodies, whereby these are free from contaminations.
Various methods for the production of anti-MIF antibodies are currently available. One major approach is to use the recombinant production of anti-MIF antibodies whereby a host cell expresses the desired anti-MIF antibody product. Chinese hamster ovary (CHO) cells are a cell line derived from the ovary of the Chinese hamster (Cricetulus griseus). They are frequently and broadly used in biological and medical research production of therapeutic proteins, e.g. antibodies.
Today, CHO cells are the most commonly used mammalian hosts for industrial production of recombinant protein therapeutics, including antibodies.
CHO cells have been a cell line of choice because of their rapid growth and high protein production. They have become the mammalian equivalent of E. coli in research and biotechnology today, especially when long-term, stable gene expression and high yields of proteins are required.
However, the present inventors, upon investigation of a possible preferable production and purification process of anti-MIF antibodies with the use of CHO cells as host cells discovered that CHO cells themselves produce MIF. This is surprisingly different from the situation e.g. when preparing MIF from hybridoma cells or in the preparation of polyclonal antisera where no such or corresponding contaminations are found. The MIF as produced by CHO cells is a Chinese hamster MIF, due to the fact that CHO cells are derived from ovary cells of a Chinese hamster. This “Chinese hamster-MIF” (in the following and above also designated as “CHO-MIF”), possibly because of the high homology between CHO-MIF and other, e.g. human, MIF also binds to the anti-MIF antibodies to be produced. Thus, endogenous CHO-MIF could possibly contaminate the final CHO-cell based preparations of antibodies directed to non-CHO-MIF (e.g. complexed to the desired anti-MIF antibodies), like e.g. human MIF, or mouse MIF.
Therefore, there exists a need for the provision of a cell line which does not produce possibly contaminating CHO-MIF; a further need exists for a sensitive method to detect minor amounts of CHO-MIF contaminations in preparations of anti-MIF antibodies produced in CHO cells producing the CHO-MIF and a specific method for the production and purification of such anti-MIF antibody preparations which are not contaminated by CHO-MIF. As a prerequisite for both the provision of an essentially CHO-MIF free CHO cell line and for developing a sensitive detection method for potential CHO-MIF contaminations, there exists a need to identify and characterize the CHO-MIF gene as a starting point for solving the problems mentioned above.
There also exists a need for such a CHO-MIF cell line which provides similar growth and production characteristics as the wild type CHO cell line.